Reactive free radicals cause protein inactivation, DNA damage, and oxidation of amino acids, lipids, and glucose. Some oxidation products become ketoaldehydes, which cause glycation of proteins found in diabetes. The glutathione (GSH) is thought to prevent this oxidation by scavenging hydroxyl and other reactive radicals which yield the glutathionyl radical. We report here the detection of endogenous intracellular glutathionyl radicals and also the identity of the free radical produced from the reaction between `-ketoaldehyde and amino acids. We used EPR and the spin trapping method with 5,5-dimethyl-1- pyrroline N-oxide (DMPO). The NCB-20 cells incubated with DMPO exhibited an EPR spectrum of glutathionyl radical adducts of DMPO upon hydrogen peroxide challenge. The identity of this radical was confirmed by observing similar spectra from the known enzyme reactions. The radical formation required viable cells and biosynthesis of GSH. N-acetyl-L-cysteine (NAC)-treated cells produced NAC-derived radicals in place of glutathionyl radicals. Glutathionyl radical formation exhibited a burst after a lag period, whereas NAC-radical formation did not show this trend. In both cases, GSH concentration decreased continuously during the stress. Therefore, it is likely that the protective mechanism of antioxidant enzymes against oxidative stress in cells became inefficient after the lag period and NAC may protect antioxidant enzyme systems against inactivation by a mechanism yet to be identified. The reaction between methylglyoxal and amino acids, as a model system, indicated that a stable free radical intermediate was formed during the crosslinking reaction. By using stable isotope-labelled compounds, we identified the structure of the radical as a cation in which the amino groups of two amino acids are crosslinked by two carbonyl groups of a methylglyoxal molecule. Although the formation of this radical did not require transition metal ions or oxygen, superoxide radical anions were produced under aerobic conditions by the reaction following radical formation. The radical sites in protein crosslinks may be more stable and could be a reactive site for the generation of a toxic superoxide radical anion over a long duration.